The present invention relates to a rapid and efficient method of separation and purification of the four species of human interleukin-1 (IL-1).
A growing body of in vitro and in vivo data strongly suggests that interleukin-1 (IL-1) plays a central hormonal role in acute and chronic inflammation. For example, IL-1 has been shown to stimulate fever, the synthesis of acute phase reactants by the liver, and the proliferation of connective tissue cells involved in fibrosis. Moreover, IL-1 can stimulate in vitro the production of prostaglandins and collagenase by chondrocytes and rheumatoid synovial cells and therefore may be responsible for the destruction of articular cartilage in rheumatoid arthritis. No other inflammatory mediator is known to have this property. Interleukin-1 has also been implicated in beneficial tissue responses, such as enhanced anti-tumor activity resulting from the stimulation of natural killer cells. This diversity of biological activities suggests that the identified human IL-1 species may have different effector cell functions. To date at least four biologically active species of human IL-1 have been identified by isoelectrofocusing with isoelectric points (pI) of about 6.8 to about 7.2 (herein denoted pI 6.8), about 5.8 to about 6.2 (herein denoted pI 6.0), about 5.3 to about 5.5 (herein denoted pI 5.4) and about 5.0 to about 5.3 (herein denoted pI 5.2) (Lachman et al., J. Supramol. Struct., (1980) 13: 457-466, Schmidt et al., J. Immunol., (1982) 128: 2177-2182). The ability to unequivocally correlate biological activity with IL-1 species levels has been forestalled by the lack of highly purified human IL-1 species. Methods of purification of human IL-1 have been described in the literature but most have not purified the individual species of IL-1. Lachman et al., J. Supramol. Struct., (1980) 13: 457-466, describe a gel filtration, isoelectric focusing and polyacrylamide gel electrophoresis separation technique that results in a partially pure IL-1 preparation. Kronheim et al., J. Exp. Med., (1985) 161: 490-502, describe a multistep purification procedure that results in a preparation containing at least three of the four species of human IL-1. Schmidt, J. Exp. Med., (1984) 160: 772-787, describes a method for the purification of human IL-1 species pI 6.8 using ion exchange high pressure liquid chromatography (HPLC) in the isocratic mode followed by size exclusion HPLC. This procedure resulted in a pure preparation of the pI 6.8 species but there are several major disadvantages to this technique. First, the isocratic separations were extremely time consuming and had limited product capacity. Second, it was necessary to repeat the isocratic anion exchange HPLC in order to remove contaminants which were not entirely removed during the first step. Third, the size exclusion HPLC resulted in dilution of the sample as well as suspension of the sample in non-lyophilizable salts and thus made it difficult to use the purified material for subsequent procedures.